Project abstract Patterns of modified cytosines (5-methylcytosine and 5-hydroxymethylcytosine) are required for the normal function of the genome, and perturbations of patterns of modified cytosine can be lethal and lead to gross changes in patterns of gene expression. Methylation patterns are grossly abnormal in many forms of human cancer. Existing methods of genome-wide methylation profiling require large amounts of DNA, overestimate methylation levels, and cannot cover the entire genome. We are developing a method wherein enzymes are used to modify sites of methylation and hydroxymethylation with chemical tags that render these sites easily detectible by nanopore sequencing by synthesis and other platforms for single molecule DNA sequencing. The result will be a comprehensive method for the detection of modified bases in human genomes with much greater sensitivity, accuracy, economy, and throughput than existing methods.